Mutation of the tumor suppressor TP53 leads to the evasion of apoptosis after radiation, underlying resistance to conventional cancer treatments, such as radiation and chemotherapy. Our research seeks to bypass these alterations through the discovery of parallel cell-death pathways. We previously discovered a novel apoptotic process, termed Chk1-suppressed (CS) pathway, which restores radiosensitivity in TP53 mutant cells after treatment with Chk1 inhibitors (Sidi et al. Cell 2008)1. Using both zebrafish and mammalian TP53-mutant models, our lab has established that the CS pathway requires the assembly of a protein complex called the PIDDosome, composed of PIDD, RAIDD, and caspase-2 (Ando et al. Mol Cell 2012)2. This proposal focuses on (1) identifying further genetic determinants predicting Chk1 inhibitor efficacy in radiotherapy and (2) discovering novel radiosensitizers against tumors with TP53 mutation. Utilizing both human cancer cells and zebrafish, we investigated the impact of cancer alterations other than mutant p53 on cellular sensitivity to CS apoptosis. We found that a mutation in PTEN, the second most commonly mutated tumor suppressor, blocks the execution of apoptosis after ionizing radiation (IR) and Chk1 inhibition (Chk1i). Furthermore, we identified the PTEN-Akt-IAP signaling axis as a candidate pathway to promote resistance to CS apoptosis. The loss of PTEN appears to inhibit the CS pathway downstream of caspase-2 activation, as HeLa cells with impaired PTEN fail to engage in apoptosis, despite an unaffected ability to cleave caspase-2 following IR+Chk1i. Our hypothesis is that one or more IAPs act downstream of Akt to directly inhibit active caspase-2. I propose to test this hypothesis using a candidate knockdown approach to determine which IAP(s), if any, is responsible for caspase-2 inhibition. The findings will be extended in vivo through both chemical and genetic studies in zebrafish. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput drug screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. A primary screen of drugs treated in combination with 15 Gy IR yielded 140 compounds capable of producing phenotypes comparable to Chk1i (i.e. >75% sick or dead fish 4 days post irradiation) in p53 mutant fish. Our secondary screen of irradiated vs non-irradiated embryos so far has identified 6 compounds that demonstrate specific radiosensitizing effects. The genetic specificity of these candidate radiosensitizers will be confirmed and prioritized by clinical relevance, proposed mechanism of action, and efficacy. The proposed targets, along with the mechanism(s) of cell death will be confirmed in cell culture and in vivo. The results of these findings may aid in rapidly benefiting cancer patients by proposing a new application for an already approved and available drug.